Systems and methods for treating cancer

ABSTRACT

Embodiments of the invention provide a method of treating cancer, the method comprising providing a subject having cancer cells, and contacting the cancer cells with a therapeutically effective amount of a G2/M checkpoint inhibitor. Embodiments of the invention also provide a method of treating cancer in a subject, the method comprising the steps of: (a) receiving a sample of the cancer cells from the subject; (b) determining if at least a portion of the sample of the cancer cells is LKB1 deficient; and (c) contacting the cancer cells with a therapeutically effective amount of a G2/M checkpoint inhibitor. Embodiments of the invention also provide a method of treating cancer in a subject, the method comprising contacting the cancer cells with a therapeutically effective amount of a Wee1 inhibitor and a therapeutically effective amount of a second pharmaceutical composition. Embodiments of the invention also provide a method of prognosing or determining the severity of cancer in a subject comprising (a) determining the presence or absence of LKB1 in the cancer cells of the subject; and (b) prognosing the severity of cancer based on the presence of LKB1 deficiency in the cancer cells of the subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/740,546, filed Dec. 28, 2017, currently pending, which is a NationalPhase of International Application No. PCT/US2015/062785, filed Nov. 25,2015, now expired, which claimed priority under 35 U.S.C. § 119(e) toU.S. Provisional Patent Application No. 62/187,442, filed Jul. 1, 2015,now expired, the contents of which are herein incorporated by referencein their entirety.

FIELD OF THE INVENTION

The present invention is generally related to systems and methods fortreating one or more forms of cancer, and particularly related tosystems and methods for treating non-small cell lung cancer, includingparticular forms of non-small cell lung cancer, including LKB1 deficientforms of non-small cell lung cancer.

BACKGROUND OF THE INVENTION

Lung cancer remains the leading cause of cancer mortality in the US andthroughout the world, (Jemal A, et al. Global cancer statistics. CACancer J Clin 61(2): 69-90 (2011), with most patients presenting withadvanced stage disease. The five-year survival rate for advanced stagenon-small cell lung cancer (NSCLC) remains below 10%, necessitating theneed for novel therapeutic strategies against advanced disease. Asignificant clinical hurdle to reduce mortality in NSCLC is thepropensity for tumor cell invasiveness and metastasis. Despitepharmacological advances for NSCLC, current treatments have limitedefficacy in metastatic disease, and the majority of patients succumb tothe overwhelming tumor burden resulting from tumor spread. Evenmolecularly targeted therapeutics such as the epidermal growth factorreceptor-tyrosine kinase inhibitors (EGFR-TKIs) fail due to tumorresistance, followed by disease progression.

Thus there exists a need in a field for improved systems and methods forthe prognosis, diagnosis or treatment of lung cancer.

SUMMARY OF THE INVENTION

Various embodiments include a method of treating a disease in a subject,comprising providing a composition comprising one more G2/M checkpointinhibitors, and administering to the subject a therapeutically effectivedosage of the composition. In another embodiment, the disease is cancer.In another embodiment, the subject has a liver kinase B1 (LKB1)deficiency. In another embodiment, the disease is adenocarcinoma. Inanother embodiment, the disease is lung cancer. In another embodiment,the lung cancer is non-small cell lung cancer. In another embodiment,the G2/M checkpoint inhibitor is a Wee1 kinase inhibitor. In anotherembodiment, the Wee1 kinase inhibitor is AZD1775. In another embodiment,the method further comprises administering to the subject a secondpharmaceutical composition. In another embodiment, the secondpharmaceutical composition comprises at least one of the following: aDNA-damaging compound and a DNA repair-inhibiting compound. In anotherembodiment, the second pharmaceutical composition comprises at least oneof cisplatin, carboplatin, pemetrexed, gemcitabine, or a combinationthereof.

Other embodiments include a method of treating a subject, comprisingobtaining a sample from the subject, assaying the sample to determine apresence or absence of a liver kinase B1 (LKB1) deficiency, andadministering a therapeutically effective dosage of compositioncomprising one or more G2/M checkpoint inhibitors based on the presenceof an LKB1 deficiency. In another embodiment, the subject hasadenocarcinoma. In another embodiment, the subject has lung cancer. Inanother embodiment, the lung cancer is non-small cell lung cancer. Inanother embodiment, the G2/M checkpoint inhibitor is a Wee1 kinaseinhibitor. In another embodiment, the Wee1 kinase inhibitor is AZD1775.In another embodiment, the method further comprises administering atherapeutically effective amount of a second pharmaceutical composition.In another embodiment, the second pharmaceutical composition comprisesat least one of the following: a DNA-damaging compound and a DNArepair-inhibiting compound. In another embodiment, the composition isadministered intravenously.

Other embodiments include a method of treating cancer in a subject,comprising determining the presence of one or more biomarkers indicativeof an abnormal LKB1 signaling, and administering a therapeuticallyeffective dosage of a composition comprising one or more cancertreatments. In another embodiment, the one or more cancer treatmentsincludes a Wee1 inhibitor. In another embodiment, the abnormal LKB1signaling includes an LKB1 deficiency. In another embodiment, the one ormore cancer treatments includes at least one of cisplatin, carboplatin,pemetrexed, gemcitabine, or a combination thereof.

Various embodiments include a method of prognosing a condition in asubject, comprising obtaining a sample from the subject, assaying thesample to determine a presence or absence of a liver kinase B1 (LKB1)deficiency, and prognosing a severe form of the condition based on thepresence of an LKB1 deficiency. In another embodiment, the condition isadenocarcinoma. In another embodiment, the condition is non-small celllung cancer.

Other embodiments include a pharmaceutical composition, comprising oneor more G2/M checkpoint inhibitors, and a pharmaceutically acceptablecarrier.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousembodiments of the invention.

DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1A illustrates, in accordance with embodiments herein, the impactof various concentrations of a G2/M checkpoint inhibitor, AZD1775, onvarious cell lines with different genetic backgrounds. LKB1 deficientcells had the greatest reduced viability.

FIG. 1B illustrates, in accordance with embodiments herein, the impactof various concentrations of a G2/M checkpoint inhibitor, AZD1775, onvarious cell lines with different genetic backgrounds. LKB1 deficientcells had the lowest EC50 values.

FIG. 2A illustrates, in accordance with embodiments herein, the role ofLKB1 in the sensitivity of A427 cells to AZD1775. The A427 cell line wastransfected with either a rescue copy of LKB1 (“LKB1”) or a copy of LKB1with a non-functional kinase domain (“KDLKB1”) that producesnon-functional LKB1. As illustrated in FIG. 2A, the cells transfectedwith the rescue copy of LKB1 were less sensitive to AZD1775, as measuredby percent viability, compared to cells that were transfected with thecontrol non-functional kinase version of LKB1.

FIG. 2B illustrates, in accordance with embodiments herein, the role ofLKB1 in the sensitivity of A427 cells to AZD1775. The A549 cell line wastransfected with either a rescue copy of LKB1 (“LKB1”) or a copy of LKB1with a non-functional kinase domain (“KDLKB1”) that producesnon-functional LKB1. As illustrated in FIG. 2B, the cells transfectedwith the rescue copy of LKB1 were less sensitive to AZD1775, as measuredby percent viability, compared to cells that were transfected with thecontrol non-functional kinase version of LKB1.

FIG. 2C illustrates, in accordance with embodiments herein, the role ofLKB1 in the sensitivity of A427 cells to AZD1775. The H2030 cell linewas transfected with either a rescue copy of LKB1 (“LKB1”) or a copy ofLKB1 with a non-functional kinase domain (“KDLKB1”) that producesnon-functional LKB1. As illustrated in FIG. 2C, the cells transfectedwith the rescue copy of LKB1 were less sensitive to AZD1775, as measuredby percent viability, compared to cells that were transfected with thecontrol non-functional kinase version of LKB1.

FIG. 2D illustrates, in accordance with embodiments herein, the role ofLKB1 in the sensitivity of murine cells to AZD1775. 3381A murine cells(which were KRas mutant and LKB1 deficient) were transfected with eithera rescue copy of LKB1 (“LKB1”) or an empty vector that did not include acopy of LKB1 (“pBabe”). FIG. 2D illustrates that the cells transfectedwith the rescue copy of LKB1 were less sensitive to AZD1775, as measuredby percent viability, compared to cells that were transfected with thecontrol empty vector.

FIG. 3A illustrates, in accordance with embodiments herein, that theviability of H2030 cells (KRas mutant and LKB1 deficient) treated withAZD1775 (also referred to as MK1775) and cisplatin, is reduced whenAZD1775 is combined with DNA-damaging compounds or DNA repair-inhibitingcompounds.

FIG. 3B illustrates, in accordance with embodiments herein, that theviability of H2030 cells (KRas mutant and LKB1 deficient) treated withAZD1775 (also referred to as MK1775) and pemetrexed, is reduced whenAZD1775 is combined with DNA-damaging compounds or DNA repair-inhibitingcompounds. H2030 cells were transfected with a rescue copy of LKB1 or aversion of LKB1 with a non-functional kinase domain (KD) that producesnon-functional LKB1 protein.

FIG. 3C illustrates, in accordance with embodiments herein, that theviability of H2030 cells (KRas mutant and LKB1 deficient) treated withAZD1775 (also referred to as MK1775) and gemcitabine, is reduced whenAZD1775 is combined with DNA-damaging compounds or DNA repair-inhibitingcompounds. H2030 cells were transfected with a rescue copy of LKB1 or aversion of LKB1 with a non-functional kinase domain (KD) that producesnon-functional LKB1 protein.

FIG. 3D illustrates, in accordance with embodiments herein, that asimilar phenomenon as FIGS. 3A-C was noted in the 3381A murine cells inthe case of cisplatin. Similar data was also noted with pemetrexed andgemcitabine.

FIG. 4A illustrates, in accordance with embodiments herein, theexamination of cell signaling molecules in A549 cells, including bothrescue and control conditions. The cells were treated with AZD1775 aloneand in combination with 2Gy of ionizing radiation. The LKB1-cellsexhibited increased amounts of phosphorylated AMPK, but theLKB1-deficient cells exhibited increased amount of phosphorylated H2AX,which is a known indicator of DNA damage.

FIG. 4B illustrates, in accordance with embodiments herein, theexamination of cell signaling molecules in H2030 cells, including bothrescue and control conditions. The cells were treated with AZD1775 aloneand in combination with 2Gy of ionizing radiation. The LKB1-cellsexhibited increased amounts of phosphorylated AMPK, but theLKB1-deficient cells exhibited increased amount of phosphorylated H2AX,which is a known indicator of DNA damage.

FIG. 5 illustrates, in accordance with embodiments herein, the effect onA549 cells of AZD1775, alone or in combination with anotherchemotherapeutic (cisplatin) at 4 hours and 24 hours.

FIG. 6 illustrates, in accordance with embodiments herein, that LKB1deficient H2030 cells exhibit increased Caspase 3 cleavage when treatedwith a combination of AZD1775 and cisplatin, with Caspase 3 cleavagebeing another indicator of the induction of apoptosis.

FIG. 7A illustrates, in accordance with embodiments herein, that thereis increased detection of cleaved Caspase 3 in A549 cells that are LKB1deficient and treated with a combination of AZD175 and gemcitabine.

FIG. 7B illustrates, in accordance with embodiments herein, that thereis increased detection of cleaved Caspase 3 in H2030 cells that are LKB1deficient and treated with a combination of AZD175 and gemcitabine.

FIG. 8 illustrates, in accordance with embodiments herein, improvedtreatment capabilities AZD1775 in combination with otherchemotherapeutics in LKB1 deficient cells. H2030 cells were divided intothree conditions (i) pBabe in which the cells were transfected with anempty vector and remained LKB1 deficient; (ii) LKB1 in which the cellswere transfected with a functioning rescue copy of LKB1; and (iii) p53in which the cells were transfected with a functioning rescue copy ofp53 (i.e., the cells functioned as wild type p53 cells). The data inFIG. 8 shows that the combination of AZD1775 and cisplatin leads tosignificantly increased amounts of cleaved PARP and cleaved Caspase 3,which both indicate an increased amount of apoptosis occurring in thispopulation of H2030 cells. Moreover, cells in the other conditions (LKB1and p53) did not exhibit the increases in cleaved PARP and Caspase 3,although the p53 cells exhibited increased cleavage of PARP and Caspase3 relative to the functional LKB1 cells.

FIG. 9 illustrates, in accordance with embodiments herein, mutantkras/lkb1 deficient nscic tumors in transgenic mice. Mice treated withAZD1775+Cisplatin survived longer than mice treated with Cisplatinalone.

DETAILED DESCRIPTION

All references, publications, and patents cited herein are incorporatedby reference in their entirety as though they are fully set forth.Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Hornyak, et al., Introductionto Nanoscience and Nanotechnology, CRC Press (2008); Singleton et al.,Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley &Sons (New York, N.Y. 2001); March, Advanced Organic Chemistry Reactions,Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, N.Y. 2013);and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4th ed.,Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2012),provide one skilled in the art with a general guide to many of the termsused in the present application. One skilled in the art will recognizemany methods and materials similar or equivalent to those describedherein, which could be used in the practice of the present invention.Indeed, the present invention is in no way limited to the methods andmaterials described.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described.

Some embodiments of the invention provide a method of treating cancercells in a subject. In some embodiments, the cancer cells are LKB1deficient and the method may include contacting the LKB1 deficientcancer cells with a therapeutically effective amount of a G2/Mcheckpoint inhibitor. In some embodiments, the cancer may comprise anadenocarcinoma, such as lung cancer. For example, the lung cancer maycomprise non-small cell lung cancer. Moreover, in some embodiments, theG2/M checkpoint inhibitor may comprise a Wee1 kinase inhibitor, such asAZD1775. In addition, in some embodiments, the method may also includecontacting the cells that are LKB1 deficient with a therapeuticallyeffective amount of a second pharmaceutical composition. In someaspects, the second pharmaceutical composition may comprise at least oneof the following: a DNA-damaging compound and a DNA repair-inhibitingcompound. In other aspects, the second pharmaceutical composition maycomprise at least one of cisplatin, carboplatin, pemetrexed,gemcitabine, and a combination thereof.

Some embodiments of the invention may include a method of treatingadenocarcinoma cells in a subject, with may include the steps of (i)receiving a sample of the adenocarcinoma cells; (ii) determining if atleast a portion of the sample of the adenocarcinoma cells is LKB1deficient; and (iii) contacting the adenocarcinoma cells with atherapeutically effective amount of a G2/M checkpoint inhibitor. In someembodiments, the adenocarcinoma is lung cancer. For example, the lungcancer may comprise non-small cell lung cancer. Moreover, in someembodiments, the G2/M checkpoint inhibitor may comprise a Wee1 kinaseinhibitor, such as AZD1775. In addition, in some embodiments, the methodmay also include contacting the cells that are LKB1 deficient with atherapeutically effective amount of a second pharmaceutical composition.In some aspects, the second pharmaceutical composition may comprise atleast one of the following: a DNA-damaging compound and a DNArepair-inhibiting compound. In some particular aspects, thetherapeutically effective amount of the second pharmaceuticalcomposition is less than an amount that would be used without the G2/Mcheckpoint inhibitor.

Some further embodiments of the invention provide a method of treatingadenocarcinoma cells in a subject. In some aspects, the adenocarcinomacells are LKB1 deficient. The methodology may include contacting theLKB1 deficient adenocarcinoma cells with a therapeutically effectiveamount of a Wee1 inhibitor (e.g., AZD1775) and contacting the LKB1deficient cancer cells with a therapeutically effective amount of asecond pharmaceutical composition. In some embodiments, theadenocarcinoma is lung cancer. For example, the lung cancer may comprisenon-small cell lung cancer. In some aspects, the second pharmaceuticalcomposition may comprise at least one of the following: a DNA-damagingcompound and a DNA repair-inhibiting compound. In other aspects, thesecond pharmaceutical composition may comprise at least one ofcisplatin, carboplatin, pemetrexed, gemcitabine, and a combinationthereof.

Some embodiments of the invention provide methods of treating a disease,such as cancer. For example, some embodiments of the invention includemethods of treating the cancer. In some aspects, the cancer may comprisea specific form of cancer, such as adenocarcinoma. In some embodiments,the adenocarcinoma may be a specific form of cancer, such as lungcancer, which may further include non-small cell lung cancer. In somespecific aspects, the methods may include detecting and/or quantifying,and/or augmenting the presence and/or activity of one or more markers.In some embodiments of the invention, the marker may comprise one ormore of LKB1, RAS (e.g., KRas), and/or Wee1. In particular, someembodiments include augmenting (e.g., increasing or decreasing) a levelof expression and/or functionality of the one or more markers and thenproviding one or more therapeutics to a patient to treat the cancer. Insome aspects, some embodiments include making therapeutic decisionsbased at least partially upon a determination of expression and/orfunctionality of one or more markers (e.g., LKB1).

The present invention can be directed to methods for treating cancer.For example, methods according to some embodiments may compriseproviding therapeutically effective amounts of one or morepharmaceutical compositions, alone or in combination, to the subjectwith cancer. In some aspects, the method may include administering oneor more pharmaceutical compositions, alone or in combination, that canfunction to inhibit one or more markers, such as Wee1. In particular,the administration of one or more pharmaceutical compositions mayinhibit one or more cellular processes such as the G2/M phase of thecell cycle. For example, a first pharmaceutical composition(s) caninhibit the G2/M phase of the cell cycle and a second composition can beadministered to the subject with cancer that can potentially induceapoptosis, necrosis, and/or cellular senescence in the cancer (e.g., aDNA-damaging composition).

Some embodiments of the invention provide methods of treating cancer ina subject, which include an assessment of one or more markers to selecta treatment. The method may include assessing an expression level of oneor more of the markers to make determinations regarding the appropriatetherapeutic decisions. For example, prior to the administration of oneor more pharmaceutical compositions, an assessment of a marker (i.e.,LKB1 and/or KRas) can be performed to determine whether a G2/Mcheckpoint inhibitor (e.g., a Wee1 kinase inhibitor) is an appropriatetherapeutic. In some aspects, the assessment of the marker may include adetermination regarding the expression level of the marker and in otheraspects, the assessment may include making a determination of thefunctionality of the gene, RNA, and/or protein version(s) of the marker.

Generally, some embodiments of the present invention can be used toidentify, quantify, detect, assess, isolate, and/or augment expressionlevels of one or more markers. A marker may be any molecular structureproduced by a cell, expressed inside the cell, accessible on the cellsurface, or secreted by the cell. A marker may be any protein,carbohydrate, fatty acid, nucleic acid, catalytic site, or anycombination of these such as an enzyme, glycoprotein, cell membrane,virus, a particular cell, or other uni- or multimolecular structure. Amarker may be represented by a sequence of a nucleic acid or any othermolecules derived from the nucleic acid. Examples of such nucleic acidsinclude miRNA, tRNA, siRNA, mRNA, cDNA, genomic DNA sequences, orcomplementary sequences thereof. Alternatively, a marker may berepresented by a protein sequence. The concept of a marker is notlimited to the exact nucleic acid sequence or protein sequence orproducts thereof, rather it encompasses all molecules that may bedetected by a method of assessing the marker. Without being limited bythe theory, the detection of the marker may encompass the detectionand/or determination of a change in copy number (e.g., copy number of agene or other forms of nucleic acid) or in the detection of one or moretranslocations.

Therefore, examples of molecules encompassed by a marker represented bya particular sequence further include alleles of the gene used as amarker. An allele includes any form of a particular nucleic acid thatmay be recognized as a form of the particular nucleic acid on account ofits location, sequence, or any other characteristic that may identify itas being a form of the particular gene. Alleles include but need not belimited to forms of a gene that include point mutations, silentmutations, deletions, frame shift mutations, single nucleotidepolymorphisms (SNPs), inversions, translocations, heterochromaticinsertions, and differentially methylated sequences relative to areference gene, whether alone or in combination.

In some embodiments of the invention, the marker may comprise aplurality of markers. For example, the plurality of markers may includeone or more molecules that are known to play a role in oncogenesisand/or the progression of cancer. In some aspects, the markers maycomprise molecules such as LKB1, Wee1, and/or a member of the Rasfamily, such as KRas. In some embodiments, a combination of one or moreof the above-described potential markers can be looked at in combinationwith other markers to provide therapeutic information for one skilled inthe art in the context of one or more cancers, for example, non-smallcell lung cancer (NSCLC).

The LKB1 tumor suppressor gene is commonly mutated in NSCLC, and offersa therapeutic opportunity. The LKB1 gene was discovered through geneticlinkage analysis of the familial disorder, Peutz-Jeghers syndrome, andhas since been found to be inactivated in 30%-50% of NSCLC patients,about twice the prevalence of EGFR mutations found in this disease.Further, convergent in vitro and in vivo studies have led to therealization that loss of LKB1 may be a critical event in NSCLC.

LKB1 (also known as STK11 serine/threonine kinase 11) is aserine-threonine kinase, phosphorylating and regulating 14 differentprotein kinases. The biological role of LKB1 regulation of these kinasesremains largely unknown except for the AMP-activated kinase, or AMPK.The primary function of LKB1-AMPK signaling is in the regulation ofcellular energy metabolism. Increases in intracellular levels of AMP,due to hypoxia, ischemia, or other stressors, induce the LKB1 dependentactivation of AMPK, allowing AMPK to alter cellular functions andrestore ATP levels within the cell.

There is a link between the loss of LKB1 and increased aggressiveness ofadenocarcinomas such as NSCLC, colorectal adenoma, prostate andendometrial adenomas. In human endometrial cancers, LKB1 expression wasfound inversely correlated with tumor grade and stage, implying thatLKB1 inactivation or down-regulation also contributes to endometrialcancer progression in women.

LKB1 is a tumor suppressor, yet somatic mutations to LKB1 appear to berare in most sporadic cancers. However, recent work has shown that inlung carcinomas, mutational loss of LKB1 occurs in about 30% to about50% of cases. NSCLC is a heterogeneous disease consisting of large cellcarcinoma (LCC), adenocarcinoma, squamous cell carcinoma (SCC) and mixedhistology tumors (adenosquamous). Among these subtypes, LKB1 lossappears to occur most frequently in adenocarcinoma (34%), with LKB1 lossoccurring in SCC (19%), LCC (14%) and adenosquamous (25%) at lowerrates.

Further, LKB1 loss synergistically cooperates with oncogenic gene KRASto decrease tumor latency and increase tumor metastasis in a transgenicmouse model of lung cancer. KRas is involved primarily in regulatingcell division. The protein relays signals from outside the cell to thecell nucleus. These signals instruct the cell to grow and divide or tomature and take on specialized functions (differentiate).

The KRas protein is a GTPase that converts GTP into GDP. The KRasprotein acts like a switch, and it is turned on and off by the GTP andGDP molecules. To transmit signals, the KRas protein must be turned onby binding to a molecule of GTP. The KRas protein is turned off(inactivated) when it converts the GTP to GDP. When the protein is boundto GDP, it does not relay signals to the cell nucleus.

Mechanistically, LKB1 functions at the center of a complex signalingnetwork, phosphorylating and activating 14 protein kinases. The bestcharacterized of the LKB1 activated kinases is the AMP-activated kinase,or AMPK. The primary function of LKB1-AMPK signaling is in theregulation of cellular energy metabolism. Increases in intracellularlevels of AMP due to hypoxia or ischemia, induce the LKB1 dependentactivation of AMPK, allowing AMPK to alter cellular functions andrestore ATP levels within the cell.

After DNA damage, cell cycle checkpoints are activated. Checkpointactivation pauses the cell cycle and gives the cell time to repair thedamage before continuing to divide. If the damage is too severe,apoptosis or senescence is induced to ensure that unrepaired DNA-damageis not passed on to future generations of cells. Loss of checkpointmechanisms is frequently found in cancer, a trait which can be exploitedin cancer therapy. Wee1 is a kinase involved in checkpoint regulationthat in response to DNA-damage or replication stress can halt the cellcycle progression in S- and G2 phases by adding inhibitoryphosphorylations (Tyr15) on cyclin-dependent kinases CDK2 and CDK1,respectively. Previous studies have found that Wee1 is up-regulated insome cancers, such as melanoma, and that high expression of Wee1 wasassociated with poor disease-free survival. Likewise, over-expression ofWee1 (protein and/or mRNA) has been reported in osteosarcoma,glioblastoma and ovarian- and vulvar squamous cell carcinomas, thusemphasizing its potential as a therapeutic target in cancer.

An allele of a gene may or may not produce a functional protein; mayproduce a protein with altered function, localization, stability,dimerization, or protein-protein interaction; may have overexpression,under-expression or no expression; may have altered temporal or spatialexpression specificity; or may have altered copy number (e.g., greateror less numbers of copies of the allele). An allele may also be called amutation or a mutant. An allele may be compared to another allele thatmay be termed a wild type form of an allele. In some cases, the wildtype allele is more common than the mutant.

Some embodiments of the invention may comprise the use of one or moremethods of amplifying a nucleic acid-based starting material (i.e., atemplate). Nucleic acids may be selectively and specifically amplifiedfrom a template nucleic acid contained in a sample. In some nucleic acidamplification methods, the copies are generated exponentially. Examplesof nucleic acid amplification methods known in the art include:polymerase chain reaction (PCR), ligase chain reaction (LCR),self-sustained sequence replication (3 SR), nucleic acid sequence basedamplification (NASBA), strand displacement amplification (SDA),amplification with Qβ replicase, whole genome amplification with enzymessuch as φ29, whole genome PCR, in vitro transcription with T7 RNApolymerase or any other RNA polymerase, or any other method by whichcopies of a desired sequence are generated.

In addition to genomic DNA, any oligonucleotide or polynucleotidesequence can be amplified with an appropriate set of primer molecules.In particular, the amplified segments created by the PCR process itselfare, themselves, efficient templates for subsequent PCR amplifications.In some embodiments, the first and/or the second reagents may compriseone or more oligonucleotides (e.g., primers) that can specifically bindto DNA, RNA, and/or cDNA to detect the presence and/or expression ofnucleic acids that correspond to one of the markers (e.g., LKB1 and/orKRas) using techniques such as PCR, qPCR, qRT-PCR, northern blot, etc.

PCR generally involves the mixing of a nucleic acid sample, two or moreprimers that are designed to recognize the template DNA, a DNApolymerase, which may be a thermostable DNA polymerase such as Taq orPfu, and deoxyribose nucleoside triphosphates (dNTP's). Reversetranscription PCR, quantitative reverse transcription PCR, andquantitative real time reverse transcription PCR are other specificexamples of PCR. In general, the reaction mixture is subjected totemperature cycles comprising a denaturation stage (typically 80-100°C.), an annealing stage with a temperature that is selected based on themelting temperature (Tm) of the primers and the degeneracy of theprimers, and an extension stage (for example 40-75° C.). In real-timePCR analysis, additional reagents, methods, optical detection systems,and devices known in the art are used that allow a measurement of themagnitude of fluorescence in proportion to concentration of amplifiedDNA. In such analyses, incorporation of fluorescent dye into theamplified strands may be detected or measured.

Alternatively, labeled probes that bind to a specific sequence duringthe annealing phase of the PCR may be used with primers. Labeled probesrelease their fluorescent tags during the extension phase so that thefluorescence level may be detected or measured. Generally, probes arecomplementary to a sequence within the target sequence downstream fromeither the upstream or downstream primer. Probes may include one or morelabel. A label may be any substance capable of aiding a machine,detector, sensor, device, or enhanced or unenhanced human eye fromdifferentiating a labeled composition from an unlabeled composition.Examples of labels include but are not limited to: a radioactive isotopeor chelate thereof, dye (fluorescent or nonfluorescent,) stain, enzyme,or nonradioactive metal. Specific examples include, but are not limitedto: fluorescein, biotin, digoxigenin, alkaline phosphatese, biotin,streptavidin, ³H, ¹⁴C, ³²P, ³⁵S, or any other compound capable ofemitting radiation, rhodamine, 4-(4′-dimethylamino-phenylazo) benzoicacid (“Dabcyl”); 4-(4′-dimethylamino-phenylazo)sulfonic acid (sulfonylchloride) (“Dabsyl”); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonicacid (“EDANS”); Psoralene derivatives, haptens, cyanines, acridines,fluorescent rhodol derivatives, cholesterol derivatives;ethylenediaminetetraaceticacid (“EDTA”) and derivatives thereof or anyother compound that may be differentially detected. The label may alsoinclude one or more fluorescent dyes optimized for use in genotyping.Examples of dyes facilitating the reading of the target amplificationinclude, but are not limited to: CAL-Fluor Red 610, CAL-Fluor Orange560, dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED,dROX, PET, BHQ+, Gold540, and LIZ.PCR facilitating the reading of thetarget amplification.

Either primers or primers along with probes allow a quantification ofthe amount of specific template DNA present in the initial sample. Inaddition, RNA may be detected by PCR analysis by first creating a DNAtemplate from RNA through a reverse transcriptase enzyme. The markerexpression may be detected by quantitative PCR analysis facilitatinggenotyping analysis of the samples.

An illustrative example, using dual-labeled oligonucleotide probes inPCR reactions is disclosed in U.S. Pat. No. 5,716,784 to DiCesare. Inone example of the PCR step of the multiplex Real Time-PCR/PCR reactionof the present invention, the dual-labeled fluorescent oligonucleotideprobe binds to the target nucleic acid between the flankingoligonucleotide primers during the annealing step of the PCR reaction.The 5′ end of the oligonucleotide probe contains the energy transferdonor fluorophore (reporter fluor) and the 3′ end contains the energytransfer acceptor fluorophore (quenching fluor). In the intactoligonucleotide probe, the 3′ quenching fluor quenches the fluorescenceof the 5′ reporter fluor. However, when the oligonucleotide probe isbound to the target nucleic acid, the 5′ to 3′ exonuclease activity ofthe DNA polymerase, e.g., Taq DNA polymerase, will effectively digestthe bound labeled oligonucleotide probe during the amplification step.Digestion of the oligonucleotide probe separates the 5′ reporter fluorfrom the blocking effect of the 3′ quenching fluor. The appearance offluorescence by the reporter fluor is detected and monitored during thereaction, and the amount of detected fluorescence is proportional to theamount of fluorescent product released. Examples of apparatus suitablefor detection include, e.g. Applied Biosystems™ 7900HT real-time PCRplatform and Roche's 480 LightCycler, the ABI Prism 7700 sequencedetector using 96-well reaction plates or GENEAMP PC System 9600 or 9700in 9600 emulation mode followed by analysis in the ABA Prism SequenceDetector or TAQMAN LS-50B PCR Detection System. The labeled probefacilitated multiplex Real Time-PCR/PCR can also be performed in otherreal-time PCR systems with multiplexing capabilities.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. Amplification may be a template-specificreplication or a non-template-specific replication (i.e., replicationmay be specific template-dependent or not). Template specificity is heredistinguished from fidelity of replication (synthesis of the properpolynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“marker” specificity. Marker sequences are “markers” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

The term “template” refers to nucleic acid originating from a samplethat is analyzed for the presence of a marker of interest. In contrast,“background template” or “control” is used in reference to nucleic acidother than sample template that may or may not be present in a sample.Background template is most often inadvertent. It may be the result ofcarryover, or it may be due to the presence of nucleic acid contaminantssought to be purified out of the sample. For example, nucleic acids fromorganisms other than those to be detected may be present as backgroundin a test sample.

In addition to primers and probes, template specificity is also achievedin some amplification techniques by the choice of enzyme. Amplificationenzymes are enzymes that, under the conditions in which they are used,will process only specific sequences of nucleic acid in a heterogeneousmixture of nucleic acid. Other nucleic acid sequences will not bereplicated by this amplification enzyme. Similarly, in the case of T7RNA polymerase, this amplification enzyme has a stringent specificityfor its own promoters (Chamberlin et al. (1970) Nature (228):227). Inthe case of T4 DNA ligase, the enzyme will not ligate the twooligonucleotides or polynucleotides, where there is a mismatch betweenthe oligonucleotide or polynucleotide substrate and the template at theligation junction (Wu and Wallace (1989) Genomics (4):560). Finally, Taqand Pfu polymerases, by virtue of their ability to function at hightemperature, are found to display high specificity for the sequencesbounded and thus defined by the primers; the high temperature results inthermodynamic conditions that favor primer hybridization with the targetsequences and not hybridization with non-target sequences (H. A. Erlich(ed.) (1989) PCR Technology, Stockton Press).

The term “amplifiable nucleic acid” refers to nucleic acids that may beamplified by any amplification method. It is contemplated that“amplifiable nucleic acid” will usually comprise “sample template.” Theterms “PCR product,” “PCR fragment,” and “amplification product” referto the resultant mixture of compounds after two or more cycles of thePCR steps of denaturation, annealing and extension. These termsencompass the case where there has been amplification of one or moresegments of one or more target sequences.

In some forms of PCR assays, quantification of a target in an unknownsample is often required. Such quantification is often in reference tothe quantity of a control sample. The control sample DNA may beco-amplified in the same tube in a multiplex assay or may be amplifiedin a separate tube. Generally, the control sample contains DNA at aknown concentration. The control sample DNA may be a plasmid constructcomprising only one copy of the amplification region to be used asquantification reference. To calculate the quantity of a target in anunknown sample, various mathematical models are established.Calculations are based on the comparison of the distinct cycledetermined by various methods, e.g., crossing points (CP) and cyclethreshold values (Ct) at a constant level of fluorescence; or CPacquisition according to established mathematic algorithm.

The algorithm for Ct values in real time-PCR calculates the cycle atwhich each PCR amplification reaches a significant threshold. Thecalculated Ct value is proportional to the number of target copiespresent in the sample, and the Ct value is a precise quantitativemeasurement of the copies of the target found in any sample. In otherwords, Ct values represent the presence of respective target that theprimer sets are designed to recognize. If the target is missing in asample, there should be no amplification in the Real Time-PCR reaction.

Alternatively, the Cp value may be utilized. A Cp value represents thecycle at which the increase of fluorescence is highest and where thelogarithmic phase of a PCR begins. The LightCycler® 480 Softwarecalculates the second derivatives of entire amplification curves anddetermines where this value is at its maximum. By using thesecond-derivative algorithm, data obtained are more reliable andreproducible, even if fluorescence is relatively low.

The various and non-limiting embodiments of the PCR-based methoddetecting marker expression level as described herein may comprise oneor more probes and/or primers. Generally, the probe or primer contains asequence complementary to a sequence specific to a region of the nucleicacid of the marker gene. A sequence having less than 60% 70%, 80%, 90%,95%, 99% or 100% identity to the identified gene sequence may also beused for probe or primer design if it is capable of binding to itscomplementary sequence of the desired target sequence in marker nucleicacid.

An oligonucleotide may be any polynucleotide of at least 2 nucleotides.Oligonucleotides may be less than 10, 15, 20, 30, 40, 50, 75, 100, 200,or 500 nucleotides in length. While oligonucleotides are often linear,they may assume a circular or other two dimensional structure.Oligonucleotides may be chemically synthesized by any of a number ofmethods including sequential synthesis, solid phase synthesis, or anyother synthesis method now known or yet to be disclosed. Alternatively,oligonucleotides may be produced by recombinant DNA based methods. Insome aspects of the invention, an oligonucleotide may be 2 to 1000 basesin length. In other aspects, it may be 5 to 500 bases in length, 5 to100 bases in length, 5 to 50 bases in length, or 10 to 30 bases inlength. One skilled in the art would understand the length ofoligonucleotide necessary to perform a particular task. Oligonucleotidesmay be directly labeled, used as primers in PCR or sequencing reactions,or bound directly to a solid substrate as in oligonucleotide arrays. Forexample, as described in greater detail herein, in some aspects of theinvention, a first reagent can be used to detect c-Met and a secondreagent can be used to detect Fn14. In some embodiments, the firstand/or the second reagents may comprise one or more oligonucleotides(e.g., primers) that can specifically bind to DNA, RNA, and/or cDNA todetect the presence and/or expression of nucleic acids that correspondto one or more markers (e.g., LKB1 and/or KRas) using techniques such asPCR, qPCR, qRT-PCR, northern blot, etc.

Some embodiments of the invention may include assessing, determining,quantifying, or altering the expression of a marker. As used hereinexpression encompasses any and all processes through which materialderived from a nucleic acid template may be produced. Expression thusincludes RNA transcription, mRNA splicing, protein translation, proteinfolding, post-translational modification, membrane transport,associations with other molecules, addition of carbohydrate moieties toproteins, phosphorylation, protein complex formation and any otherprocess along a continuum that results in biological material derivedfrom genetic material. Expression also encompasses all processes throughwhich the production of material derived from a nucleic acid templatemay be actively or passively suppressed. Such processes include allaspects of transcriptional and translational regulation. Examplesinclude heterochromatic silencing, transcription factor inhibition, anyform of RNAi silencing, microRNA silencing, small interfering RNAsilencing, alternative splicing, protease digestion, posttranslationalmodification, and alternative protein folding.

Expression may be assessed by any number of methods used to detectmaterial derived from a nucleic acid template used currently in the artand yet to be developed. Examples of such methods include any nucleicacid detection method including the following non-limiting examples,microarray analysis, RNA in situ hybridization, RNAse protection assay,Northern blot, reverse transcriptase PCR, quantitative PCR, quantitativereverse transcriptase PCR, quantitative real-time reverse transcriptasePCR, reverse transcriptase treatment followed by direct sequencing, orany other method of detecting a specific nucleic acid now known or yetto be disclosed. Other examples include any process of assessingexpression that uses an antibody including the following non-limitingexamples, flow cytometry, immunohistochemistry, ELISA, Western blot, andimmunoaffinity chromatography. Antibodies may be monoclonal, polyclonal,or any antibody fragment including an Fab, F(ab)₂, Fv, scFv, phagedisplay antibody, peptibody, multispecific ligand, or any other reagentwith specific binding to a marker. Such methods also include directmethods used to assess protein expression including the followingnonlimiting examples: HPLC, mass spectrometry, protein microarrayanalysis, PAGE analysis, isoelectric focusing, 2-D gel electrophoresis,and enzymatic assays. For example, as described in greater detailherein, in some aspects of the invention, a first reagent can be used todetect c-Met and a second reagent can be used to detect Fn14. In someembodiments, the first and/or the second reagents may comprise one ormore antibodies that can specifically bind to protein to detect thepresence and/or expression of proteins that correspond to the markers(e.g., LKB1 and/or KRas). For example, the first and second reagents inthe protein context can be assessed using techniques such asimmunohistochemistry, western blot analysis, flow cytometry, ELISA, andimmunoaffinity chromatography. Samples from which expression may bedetected include single cells, whole organs or any fraction of a wholeorgan, whether in vitro, ex vivo, in vivo, or post-mortem.

Other methods used to assess expression include the use of natural orartificial ligands capable of specifically binding one or more markers,including a protein, carbohydrate, fat, nucleic acid, catalytic site, orany combination of these such as an enzyme, glycoprotein, cell membrane,virus, cell, organ, organelle, or any uni- or multimolecular structurethat constitutes a marker that may be specifically bound by a ligand.Such ligands include antibodies, antibody complexes, conjugates, naturalligands, small molecules, nanoparticles, or any other molecular entitycapable of specific binding to a marker. Ligands may be associated witha label such as a radioactive isotope or chelate thereof, dye(fluorescent or non-fluorescent,) stain, enzyme, metal, or any othersubstance capable of aiding a machine or a human eye fromdifferentiating a cell expressing a marker from a cell not expressing amarker. Additionally, expression may be assessed by monomeric ormultimeric ligands associated with substances capable of killing thecell. Such substances include protein or small molecule toxins,cytokines, pro-apoptotic substances, pore forming substances,radioactive isotopes, or any other substance capable of killing a cell.

Positive expression encompasses any difference between a cell expressingmarkers and a cell that does not express one or more of the markers. Theexact nature of positive expression varies by the method, but is wellknown to those skilled in the art of practicing a particular method.Positive expression may be assessed by a detector, an instrumentcontaining a detector, or by aided or unaided human eye. Examplesinclude but are not limited to specific staining of cells expressing atarget in an IHC slide, binding of RNA from a sample to a microarray anddetection of binding through the use of said microarray, a particularrate of dye incorporation in real-time RTPCR measured in ΔCt oralternatively in the number of PCR cycles necessary to reach aparticular optical density at a wavelength at which a double strandedDNA binding dye (e.g. SYBR Green) incorporates, through release of labelfrom a previously labeled reporter probe used in a real-time RTPCRreaction, detection of fluorescence on a cell expressing a target by aflow cytometer, the presence of radiolabeled bands on film in a Northernblot, detection of labeled blocked RNA by RNAse protection assay, celldeath measured by apoptotic markers, cell death measured by shrinkage ofa tumor, or any other signal for the expression of a marker in existencenow or yet to be developed. In some aspects of the invention, positiveexpression is a sufficient level of expression to correlate with aparticular response such as susceptibility to cancer recurrence.

In some aspects of the invention, reduced expression constitutes nodetectable expression. However, the concept of reduced expressionfurther encompasses insufficient expression to reach or exceed athreshold, cutoff, or level that has been previously shown to result ina particular cellular or physiological response. Reduced expression mayinclude similar expression relative to a control that has beenpreviously determined not to express the marker(s) or similar expressionto a control that has been previously determined not to exhibit theresponse. In this case, even though expression may be detectable, itstill constitutes reduced expression. In some aspects of the invention,an expression level of a marker in a control known to have a reduced orincrease risk of recurrence is predetermined and expression similar tothat level is correlated with reduced or increase risk of recurrence.Increased or reduced expression includes expression that is 75% 50%,25%, 10%, 5%, 1%, 0.1%, greater or less of that of a control cell or amedian level of expression in a population. Reduced expression may alsoinclude greater than or less than 1×10⁻⁵ greater or less expressionnormalized to the expression of a housekeeping gene.

The invention contemplates assessing the expression of the marker(s) inany biological sample from which the expression may be assessed. Oneskilled in the art would know to select a particular biological sampleand how to collect said sample depending upon the marker that is beingassessed. Examples of sources of samples include but are not limited tobiopsy or other in vivo or ex vivo analysis of prostate, breast, skin,muscle, fascia, brain, endometrium, lung, head and neck, pancreas, smallintestine, blood, liver, testes, ovaries, colon, skin, stomach,esophagus, spleen, lymph node, bone marrow, kidney, placenta, or fetus.In some aspects of the invention, the sample comprises a fluid sample,such as peripheral blood, lymph fluid, ascites, serous fluid, pleuraleffusion, sputum, cerebrospinal fluid, amniotic fluid, lacrimal fluid,stool, or urine. In one aspect of the invention, the sample comprisesprimary or metastatic NSCLC cells. In another, the sample comprisessputum. In another aspect of the invention, the sample comprises bloodor other tissues obtained from a subject who has been diagnosed with oris suspected of having NSCLC.

Assessing the risk of a particular disease outcome includes theperforming of any type of test, assay, examination, result, readout, orinterpretation that correlates with an increased or decreasedprobability that an individual has had, currently has, or will develop aparticular disease, disorder, symptom, syndrome, or any conditionrelated to health or bodily state. Examples of disease outcomes include,but need not be limited to survival, death, progression of existingdisease, remission of existing disease, initiation of onset of a diseasein an otherwise disease-free subject, or the continued lack of diseasein a subject in which there has been a remission of disease. Assessingthe risk of a particular disease encompasses diagnosis in which the typeof disease afflicting a subject is determined. Assessing the risk of adisease outcome also encompasses the concept of prognosis. A prognosismay be any assessment of the risk of disease outcome in an individual inwhich a particular disease has been diagnosed. Assessing the riskfurther encompasses prediction of therapeutic response in which atreatment regimen is chosen based on the assessment. Assessing the riskalso encompasses a prediction of overall survival after diagnosis.

The sample in this method is preferably a biological sample from asubject. The term “sample” or “biological sample” is used in itsbroadest sense. Depending upon the embodiment of the invention, forexample, a sample may comprise a bodily fluid including whole blood,serum, plasma, urine, saliva, cerebral spinal fluid, semen, vaginalfluid, pulmonary fluid, tears, perspiration, mucus and the like; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to asubstrate; a tissue; a tissue print, or any other material isolated inwhole or in part from a living subject. Biological samples may alsoinclude sections of tissues such as biopsy and autopsy samples, andfrozen sections taken for histologic purposes such as blood, plasma,serum, sputum, stool, tears, mucus, hair, skin, and the like. Biologicalsamples also include explants and primary and/or transformed cellcultures derived from patient tissues.

The term “subject” is used in its broadest sense. In a preferredembodiment, the subject is a mammal. Non-limiting examples of mammalsinclude humans, dogs, cats, horses, cows, sheep, goats, and pigs.Preferably, a subject includes any human or non-human mammal, includingfor example: a primate, cow, horse, pig, sheep, goat, dog, cat, orrodent, capable of developing cancer including human patients that aresuspected of having cancer, that have been diagnosed with cancer, orthat have a family history of cancer.

Some embodiments of the invention may include a method of comparing amarker in a sample relative to one or more control samples. A controlmay be any sample with a previously determined level of expression. Acontrol may comprise material within the sample or material from sourcesother than the sample. Alternatively, the expression of a marker in asample may be compared to a control that has a level of expressionpredetermined to signal or not signal a cellular or physiologicalcharacteristic. This level of expression may be derived from a singlesource of material including the sample itself or from a set of sources.

Cancer cells include any cells derived from a tumor, neoplasm, cancer,precancer, cell line, malignancy, or any other source of cells that havethe potential to expand and grow to an unlimited degree. Cancer cellsmay be derived from naturally occurring sources or may be artificiallycreated. Cancer cells may also be capable of invasion into other tissuesand metastasis. Cancer cells further encompass any malignant cells thathave invaded other tissues and/or metastasized. One or more cancer cellsin the context of an organism may also be called a cancer, tumor,neoplasm, growth, malignancy, or any other term used in the art todescribe cells in a cancerous state.

Examples of cancers that could serve as sources of cancer cells includesolid tumors such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio sarcoma, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncancer, colorectal cancer, kidney cancer, pancreatic cancer, bonecancer, breast cancer, ovarian cancer, prostate cancer, esophagealcancer, stomach cancer, oral cancer, nasal cancer, throat cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, uterine cancer, testicular cancer, small cell lungcarcinoma, bladder carcinoma, lung cancer, epithelial carcinoma, glioma,glioblastoma multiforme, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, skin cancer, melanoma,neuroblastoma, and retinoblastoma.

Additional cancers that may serve as sources of cancer cells includeblood borne cancer, such as acute lymphoblastic leukemia (“ALL,”), acutelymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,acute myeloblastic leukemia (“AML”), acute promyelocytic leukemia(“APL”), acute monoblastic leukemia, acute erythroleukemic leukemia,acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia (“CML”), chronic lymphocytic leukemia (“CLL”), hairycell leukemia, multiple myeloma, lymphoblastic leukemia, myelogenousleukemia, lymphocytic leukemia, myelocytic leukemia, Hodgkin's disease,non-Hodgkin's Lymphoma, Waldenstrom's macroglobulinemia, Heavy chaindisease, and Polycythemia vera.

In some aspects of the invention, the cancer cells are derived fromNSCLC, which comprises any carcinoma derived from lung tissues that doesnot include small cell lung cancers. Examples of non-small cell lungcancers include adenocarcinomas, large cell carcinomas, and squamouscell carcinomas of the lung.

The pathologic stages of non-small cell lung cancer include, but are notlimited to the following: in the occult or hidden stage, cancer cellsmay be found in sputum, but no tumor can be found in the lung bybronchoscopy or other imaging. In Stage 0, also called carcinoma insitu, abnormal cells are found in the innermost lining of the lung. Suchabnormal cells are precancerous and may or may not become malignant andspread into nearby tissue.

In Stage I, a cancer has developed. There are two substages to stage 1.In Stage IA, the tumor presents only in the lung only and is 3centimeters or smaller. For the disease to be considered stage 1B, itwill have one or more of the following traits: the tumor is larger than3 centimeters, the cancer has spread to the main bronchus of the lung,and is at least 2 centimeters from the carina, the cancer has spread tothe innermost layer of the membrane that covers the lungs, or the tumorpartly blocks the bronchus or bronchioles and part of the lung hascollapsed or developed pneumonitis (inflammation of the lung).

Similarly, there are two substages to Stage II. In Stage IIA, the tumoris 3 centimeters or smaller and cancer has spread to nearby lymph nodeson the same side of the chest as the tumor. For the disease to beconsidered, Stage IIB, the cancer has spread to nearby lymph nodes onthe same side of the chest as the tumor and it will have one or more ofthe following traits: the tumor is larger than 3 centimeters, the cancerhas spread to the main bronchus of the lung and is 2 centimeters or morefrom the carina, the cancer has spread to the innermost layer of themembrane that covers the lungs, or the tumor partly blocks the bronchusor bronchioles and part of the lung has collapsed or developedpneumonitis (inflammation of the lung). Alternatively, the disease maybe classified as Stage 2B if the cancer has not spread to the lymphnodes and it displays one or more of the following traits: cancer hasspread to the chest wall, or the diaphragm, or the pleura between thelungs, or membranes surrounding the heart, the cancer has spread to themain bronchus of the lung and is no more than 2 centimeters from thecarina, but has not spread to the trachea, cancer blocks the bronchus orbronchioles and the whole lung has collapsed or developed pneumonitis(inflammation of the lung). Stage III is also divided into twosubstages.

In stage IIIA, cancer has spread to lymph nodes on the same side of thechest as the tumor and it displays one or more of the following traits:cancer has spread to the main bronchus, the chest wall, the diaphragm,the pleura around the lungs, or the membrane around the heart, but hasnot spread to the trachea, or part or all of the lung may have collapsedor developed pneumonitis (inflammation of the lung). In stage IIIB, thetumor has spread to one or more of the following: lymph nodes above thecollarbone or in the opposite side of the chest from the tumor, to theheart, to major blood vessels that lead to or from the heart, to thechest wall, to the diaphragm, to the trachea, to the esophagus, to thesternum or spine, to more than one area in the same lobe of the lung, orto the fluid of the pleural cavity surrounding the lung.

In stage IV, cancer may have spread to lymph nodes and has spread toanother lobe of the lung or to other parts of the body, such as thebrain, liver, adrenal glands, kidneys, or bone.

The present invention further provides kits to be used in assessing theexpression of a marker in a subject to assess the risk of developingdisease, diagnosing the subject as having a stage of the disease, ordetermining to which stage the disease has progressed. Kits include anycombination of components that facilitates the performance of an assay.A kit that facilitates assessing the expression of the markers mayinclude suitable nucleic acid-based and immunological reagents as wellas suitable buffers, control reagents, and printed protocols.

Kits that facilitate nucleic acid based methods may further include oneor more of the following: specific nucleic acids such asoligonucleotides, labeling reagents, enzymes including PCR amplificationreagents such as Taq or Pfu, reverse transcriptase, or other, and/orreagents that facilitate hybridization, as previously described.

In some aspects of the invention, a probe may be affixed to a solidsubstrate. In other aspects of the invention, the sample may be affixedto a solid substrate. A probe or sample may be covalently bound to thesubstrate or it may be bound by some non-covalent interaction includingelectrostatic, hydrophobic, hydrogen bonding, Van Der Waals, magnetic,or any other interaction by which a probe such as an oligonucleotideprobe may be attached to a substrate while maintaining its ability torecognize the allele to which it has specificity. A substrate may be anysolid or semi-solid material onto which a probe may be affixed, attachedor printed, either singly or in the formation of a microarray. Examplesof substrate materials include but are not limited to polyvinyl,polystyrene, polypropylene, polyester or any other plastic, glass,silicon dioxide or other silanes, hydrogels, gold, platinum, microbeads,micelles and other lipid formations, nitrocellulose, or nylon membranes.The substrate may take any form, including a spherical bead or flatsurface. For example, the probe may be bound to a substrate in the caseof an array. The sample may be bound to a substrate in the case of aSouthern Blot.

As used herein “LKB1 deficient”, an “LKB1 deficient patient,” an “LKB1deficient subject,” and/or an “LKB1 deficiency” (all usedinterchangeably) refer to either (i) an inactive and/or reduced activityin the DNA, RNA, and/or protein states of LKB1 or (ii) reduced ornon-detectable expression (i.e., at the transcriptional or translationallevel) of LKB1 such normal cellular processes that depend on LKB1 arenot functioning as intended. In other words, an LKB1 deficiency is onein which LKB1 is either not sufficiently expressed at the RNA or proteinlevel for normal cellular functioning or there is an alteration in theDNA, RNA, or amino acid sequence of LKB1 such that the resultingmolecule (if any) does not properly function. Moreover, in someembodiments, LKB1 deficiency may result from a change at the DNA or RNAlevel such that normal cellular processes such as transcription ortranslation do not properly occur or do not give rise to a functionalLKB1 molecule. In some embodiments, the LKB1 deficiency may be at leastpartially due to mutation (e.g., change in one or more synonymous and/ornon-synonymous DNA bases in the LKB1 gene), deletion of some or all ofthe LKB1 DNA sequence on one or both chromosomes, loss ofheterozygosity, and/or a specific and/or general loss of proteinexpression.

As used herein “G2/M checkpoint inhibitor” refers to any compound,molecule, structure, or other composition that is now known or laterdiscovered that can function to inhibit the G2/M checkpoint. The G2/Mcheckpoint is known to those skilled in the art as the checkpointthrough which all non-terminally differentiated cells must pass prior toentering mitosis. In some organisms, this checkpoint can be the point atwhich the cells survey DNA damage and cell size and can be controlledthrough by various molecules, such as cyclin-dependent kinases and othermolecules, such as Wee1 kinase. G2/M checkpoint inhibitors can compriseany composition that is known to at least partially initiate arrest atthe G2/M checkpoint. In some embodiments, the arrest at the G2/Mcheckpoint may be at least partially due the presence of damage to theDNA of the cell. Some examples of G2/M checkpoint inhibitors mayinclude, but are not limited to Wee1 kinase inhibitors, Chk inhibitors,aurora kinase inhibitors, and PLK inhibitors. In some particularembodiments, the G2/M checkpoint inhibitor comprises a Wee1 kinaseinhibitor.

As used herein “Wee1 kinase inhibitor” or “Wee1 inhibitor” refer to anycompound, molecule, structure, or other composition that is now known orlater discovered that can function to inhibit Wee1 kinase in an in vivoor an in vitro setting. In some aspects, the Wee1 inhibitor refers to acompound or pharmaceutical composition that is capable of inhibiting theexpression and/or the activity of the Wee1 protein or RNA. For example,a Wee1 inhibitor may comprise a compound or composition that is capableof partially or completely interrupting the physical, chemical,electrostatic or any other interactions between Wee1 protein and anyother molecules (e.g., other proteins) to inhibit the G2/M checkpoint.In particular, a Wee1 inhibitor may prevent, preclude, and/or reduce theability of Wee1, a tyrosine kinase, to phosphorylates cyclin-dependentkinase 1 (CDC2) to inactivate the CDC2/cyclin B complex, which leads toinhibition of the G2/M checkpoint. In some aspects, a Wee1 inhibitor mayinhibit or substantially reduce Wee1 expression at the transcriptionaland/or translational level. For example, the Wee1 inhibitor may comprisesiRNA, shRNA, and/or antibodies that target the Wee1 RNA or amino acidsequences. Some further non-limiting examples of Wee1 inhibitors includeAZD1775/MK-1775 and Kruppel-like factor 2.

As used herein “DNA-damaging compound” refer to any compound, molecule,structure, or other composition that is now known or later discoveredthat can function to damage the DNA of a subject/patient. DNA-damagingcompounds can include platinum-derived pharmaceutical (e.g., cisplatin,carboplatin, oxaliplatin etc.), methotrexate, doxorubicin, daunorubicinor the administration of radiation (e.g., ionizing radiation). The abovelisted agents are only illustrative in nature and any other agents canbe used that exhibit DNA-damaging properties.

As used herein “DNA repair-inhibiting compound” refer to any compound,molecule, structure, or other composition that is now known or laterdiscovered that can function to inhibit the repair of any damage to DNA.DNA repair-inhibiting compounds can include pemetrexed, gemcitabine,methylating agents (e.g., temozolomide, dacarbazine and carmustine),Alkylade®, Patrin, etc. The above listed agents are only illustrative innature and any other agents can be used that exhibit DNArepair-inhibiting properties.

Some embodiments of the invention may include the administration of apharmaceutical composition or a pharmacological composition to a subjectthat has been diagnosed with cancer. Such pharmaceutical compositionsmay take any physical form necessary depending on a number of factorsincluding the desired method of administration and the physicochemicaland stereochemical form taken by the compound or pharmaceuticallyacceptable salts of the compound. Such physical forms include a solid,liquid, gas, sol, gel, aerosol, or any other physical form now known oryet to be disclosed.

The concept of a pharmaceutical composition encompasses a compound or apharmaceutically acceptable salt thereof with or without any otheradditive. The physical form of the invention may affect the route ofadministration and one skilled in the art would know to choose a routeof administration that takes into consideration both the physical formof the compound and the disorder to be treated. Pharmaceuticalcompositions may be prepared using methodology well known in thepharmaceutical art.

In some aspects of the invention, the pharmaceutical composition cancomprise one or more compounds or products that are capable of treatinga subject with NSCLC (e.g., the treatments considered by the medicalcommunity as the “standard of care”). In some embodiments, thepharmaceutical composition may comprise or include one or more compoundsthat are capable of affecting the markers (e.g., Wee1). Thepharmaceutical composition may comprise one or more compounds that arecapable of augmenting the expression of one or more of the markers. Forexample, the pharmaceutical composition may comprise one or morecompounds that are capable of reducing expression of one or more of themarkers. In some aspects, the one or more compounds can reduce thetranscription, translation, and/or post-translational processesassociated generally or specifically with one or more of the markers.Moreover, in some embodiments, the pharmaceutical composition maycomprise one or more compounds that can inhibit the functionality of oneor more of the markers, such as Wee1.

Moreover, in some embodiments, as described in greater detail herein,the method may include the administration of a combination ofpharmaceutical compositions in subjects that may be LKB1 deficient.Specifically, in some aspects, the combination may comprise theadministration of a first pharmaceutical composition that inhibits oneof the markers (e.g., Wee1) and a second pharmaceutical composition thatcan function as conventional chemotherapeutic agent (e.g., aDNA-damaging compound or a DNA repair-inhibiting compound). Moreover, insome specific embodiments, the second pharmaceutical composition maycomprise a lesser concentration of the conventional chemotherapeuticagent(s), at least in part due to the activity of the firstpharmaceutical composition, relative to conditions that do not includethe administration of the first pharmaceutical composition.

In addition, in some aspects, one or more treatments can be provided inthe event of the detection of one or more of the markers. By way ofexample only, in some aspects, detection of reduced expression and/orreduced/eliminated functionality of one or more of the markers (e.g.,LKB1) can indicate that the subject should receive a G2/M checkpointinhibitor (e.g., a Wee1 kinase inhibitor) possibly in combination with asecond pharmaceutical composition (e.g., a DNA repair-inhibitingcompound and/or a DNA-damaging compound).

Pharmaceutical compositions include materials capable of modifying thephysical form of a dosage unit. In one non-limiting example, thecomposition includes a material that forms a coating that contains thecompound. Materials that may be used in a coating, include, for example,sugar, shellac, gelatin, or any other inert coating agent.

Pharmaceutical compositions including the disclosed agents may beprepared as a gas or aerosol. Aerosols encompass a variety of systemsincluding colloids and pressurized packages. Delivery of a compositionin this form may include propulsion of a pharmaceutical compositionincluding the disclosed compound through use of liquefied gas or othercompressed gas or by a suitable pump system. Aerosols may be deliveredin single phase, bi-phasic, or multi-phasic systems.

In some aspects of the invention, the pharmaceutical compositionincluding the disclosed agents is in the form of a solvate. Suchsolvates are produced by the dissolution of the disclosed compound in apharmaceutically acceptable solvent. Pharmaceutically acceptablesolvents include any mixtures of one or more solvents. Such solvents mayinclude pyridine, chloroform, propan-1-ol, ethyl oleate, ethyl lactate,ethylene oxide, water, ethanol, and any other solvent that delivers asufficient quantity of the disclosed compound to treat the indicatedcondition.

Pharmaceutical compositions may also include at least onepharmaceutically acceptable carrier. Carriers include any substance thatmay be administered with the disclosed compound with the intendedpurpose of facilitating, assisting, or helping the administration orother delivery of the compound. Carriers include any liquid, solid,semisolid, gel, aerosol or anything else that may be combined with thedisclosed compound to aid in its administration. Examples includediluents, adjuvants, excipients, water, and oils (including petroleum,animal, vegetable or synthetic oils.) Such carriers include particulatessuch as a tablet or powder, liquids such as oral syrup or injectableliquid, and inhalable aerosols. Further examples include saline, gumacacia, gelatin, starch paste, talc, keratin, colloidal silica, andurea. Such carriers may further include binders such as ethyl cellulose,carboxymethylcellulose, microcrystalline cellulose, or gelatin;excipients such as starch, lactose or dextrins; disintegrating agentssuch as alginic acid, sodium alginate, Primogel, and corn starch;lubricants such as magnesium stearate or Sterotex; glidants such ascolloidal silicon dioxide; sweetening agents such as sucrose orsaccharin, a flavoring agent such as peppermint, methyl salicylate ororange flavoring, or coloring agents. Further examples of carriersinclude polyethylene glycol, cyclodextrin, oils, or any other similarliquid carrier that may be formulated into a capsule. Still furtherexamples of carriers include sterile diluents such as water forinjection, saline solution, physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides, polyethylene glycols, glycerin, cyclodextrin, propyleneglycol or other solvents; antibacterial agents such as benzyl alcohol ormethyl paraben; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose, thickening agents,lubricating agents, and coloring agents.

The pharmaceutical composition may take any of a number of formulationsdepending on the physicochemical form of the composition and the type ofadministration. Such forms include solutions, suspensions, emulsions,tablets, pills, pellets, capsules, capsules including liquids, powders,sustained-release formulations, directed release formulations,lyophylates, suppositories, emulsions, aerosols, sprays, granules,powders, syrups, elixirs, or any other formulation now known or yet tobe disclosed. Additional examples of suitable pharmaceutical carriersand formulations are well known in the art.

Methods of administration include, but are not limited to, oraladministration and parenteral administration. Parenteral administrationincludes, but is not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,sublingual, intranasal, intracerebral, intraventricular, intrathecal,intravaginal, transdermal, rectal, by inhalation, or topically to theears, nose, eyes, or skin. Other methods of administration include butare not limited to infusion techniques including infusion or bolusinjection, by absorption through epithelial or mucocutaneous liningssuch as oral mucosa, rectal and intestinal mucosa. Compositions forparenteral administration may be enclosed in ampoule, a disposablesyringe or a multiple-dose vial made of glass, plastic or othermaterial.

Administration may be systemic or local. Local administration isadministration of the disclosed compound to the area in need oftreatment. Examples include local infusion during surgery; topicalapplication, by local injection; by a catheter; by a suppository; or byan implant. Administration may be by direct injection into the centralnervous system by any suitable route, including intraventricular andintrathecal injection. Intraventricular injection can be facilitated byan intraventricular catheter, for example, attached to a reservoir, suchas an Ommaya reservoir. Pulmonary administration may be achieved by anyof a number of methods known in the art. Examples include the use of aninhaler or nebulizer, formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Compoundsmay be delivered in the context of a vesicle such as a liposome or anyother natural or synthetic vesicle. Additional examples of suitablemodes of administration are well known in the art.

A pharmaceutical composition formulated to be administered by injectionmay be prepared by dissolving the disclosed compound with water so as toform a solution. In addition, a surfactant may be added to facilitatethe formation of a homogeneous solution or suspension. Surfactantsinclude any complex capable of non-covalent interaction with thedisclosed compound so as to facilitate dissolution or homogeneoussuspension of the compound.

Pharmaceutical compositions may be prepared in a form that facilitatestopical or transdermal administration. Such preparations may be in theform of a solution, emulsion, ointment, gel base, transdermal patch oriontophoresis device. Examples of bases used in such compositionsinclude petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil,diluents such as water and alcohol, and emulsifiers and stabilizers,thickening agents, or any other suitable base now known or yet to bedisclosed.

Determination of an effective and/or therapeutic amount of the disclosedagents is within the capability of those skilled in the art, especiallyin light of the detailed disclosure provided herein. The effectiveamount of a pharmaceutical composition used to affect a particularpurpose as well as its toxicity, excretion, and overall tolerance may bedetermined in vitro, or in vivo, by pharmaceutical and toxicologicalprocedures either known now by those skilled in the art or by anysimilar method yet to be disclosed. One example is the in vitrodetermination of the IC₅₀ (half maximal inhibitory concentration) of thepharmaceutical composition in cell lines or target molecules. Anotherexample is the in vivo determination of the LD₅₀ (lethal dose causingdeath in 50% of the tested animals) of the pharmaceutical composition.The exact techniques used in determining an effective amount will dependon factors such as the type and physical/chemical properties of thepharmaceutical composition, the property being tested, and whether thetest is to be performed in vitro or in vivo. The determination of aneffective amount of a pharmaceutical composition will be well known toone of skill in the art who will use data obtained from any tests inmaking that determination. Determination of an effective amount ofdisclosed compound for administration also includes the determination ofan effective therapeutic amount and a pharmaceutically acceptable dose,including the formulation of an effective dose range for use in vivo,including in humans.

As described above, in some embodiments, the administration of a firstpharmaceutical composition that may comprise a G2/M checkpointinhibitor, such as a Wee1 kinase inhibitor, may alter that effectiveamount of a second pharmaceutical composition (e.g., a DNArepair-inhibiting compound and/or a DNA-damaging compound) that may benecessary to treat the subject. For example, in some aspects, thecombination of the Wee1 inhibitor and the second pharmaceuticalcomposition may provide synergistic effects such that a reducedconcentration of the second pharmaceutical composition is needed toprovide the same therapeutic benefits as in conditions in which thefirst pharmaceutical composition is omitted. As such, in theseembodiments, there may be reduced side effects observed in the subject.

Treatment of a condition or disease is the practice of any method,process, or procedure with the intent of halting, inhibiting, slowing orreversing the progression of a disease, disorder or condition,substantially ameliorating clinical symptoms of a disease disorder orcondition, or substantially preventing the appearance of clinicalsymptoms of a disease, disorder or condition, up to and includingreturning the diseased entity to its condition prior to the developmentof the disease. Generally, the effectiveness of treatment is determinedby comparing treated groups with non-treated groups.

The addition of a therapeutically effective amount of a compoundencompasses any method of dosing of a compound. Dosing of the disclosedcompound may include single or multiple administrations of any of anumber of pharmaceutical compositions that include the disclosedcompound as an active ingredient. Examples include a singleadministration of a slow release composition, a course of treatmentinvolving several treatments on a regular or irregular basis, multipleadministrations for a period of time until a diminution of the diseasestate is achieved, preventative treatments applied prior to theinstigation of symptoms, or any other dosing regimen known in the art oryet to be disclosed that one skilled in the art would recognize as apotentially effective regimen. A dosing regimen including the regularityof and mode of administration will be dependent on any of a number offactors including but not limited to the subject being treated; theseverity of the condition; the manner of administration, the stage ofdisease development, the presence of one or more other conditions suchas pregnancy, infancy, or the presence of one or more additionaldiseases; or any other factor now known or yet to be disclosed thataffects the choice of the mode of administration, the dose to beadministered and the time period over which the dose is administered.

Pharmaceutical compositions may be administered prior to, concurrentlywith, or after administration of additional or the second pharmaceuticalcompositions. Concurrent administration means compositions areadministered within about one minute of each other. If not administeredconcurrently, the additional or second pharmaceutical compositions maybe administered a period of one or more minutes, hours, days, weeks, ormonths before or after the pharmaceutical composition that includes thecurrently disclosed compound. Alternatively, a combination ofpharmaceutical compositions may be cyclically administered. Cyclingtherapy involves the administration of one or more pharmaceuticalcompositions for a period of time, followed by the administration of oneor more different pharmaceutical compositions for a period of time andrepeating this sequential administration. Cycling therapy may be used,for example, to reduce the development of resistance to one or more ofthe compositions, to avoid or reduce the side effects of one or more ofthe compositions, and/or to improve the efficacy of the treatment.

The invention further encompasses kits that facilitate theadministration of the disclosed compound to a diseased entity. Anexample of such a kit includes one or more unit dosages of one or moreactive ingredients. The unit dosage would be enclosed in a preferablysterile container and would be comprised of the compound and apharmaceutically acceptable carrier. In another aspect, the unit dosagewould comprise one or more lyophilates of the compound. In this aspectof the invention, the kit may include another preferably sterilecontainer enclosing a solution capable of dissolving the lyophilate.However, such a solution need not be included in the kit and may beobtained separately from the lyophilate. In another aspect, the kit mayinclude one or more devices used in administrating the unit dosages or apharmaceutical composition to be used in combination with the compound.Examples of such devices include, but are not limited to, a syringe, adrip bag, a patch or an enema. In some aspects of the invention, thedevice comprises the container that encloses the unit dosage. In anotheraspect, the kit may include one or more additional compounds foradministration and administration instructions therefor.

Overall, some embodiments of the invention include systems and methodsfor treating a condition. Particular embodiments comprise the use of oneor more markers (e.g., LKB1 and/or KRAS) to make a selection of one ormore pharmaceutical compositions. In some aspects, the cancer maycomprise an adenocarcinoma, such as non-small cell lung cancer. Forexample, the method of treatment may comprise altering the expressionand/or function of one or more markers (e.g., Wee1 kinase).

As used herein, the term “G2/M checkpoint” refers to a cell cyclecheckpoint in eukaryotic cells which ensure proper division of the cell.Currently, there are three known checkpoints: the G1 checkpoint; theG2/M checkpoint; and the metaphase checkpoint, also known as the spindlecheckpoint. Each checkpoint serves as a potential halting point alongthe cell cycle, during which the conditions of the cell are assessed,with progression through the various phases of the cell cycle occurringwhen favorable conditions are met.

Following the decision to enter the cell cycle and undergo division, thecell goes through S phase, in which it replicates its DNA, and, in mostspecies, G2, in which it undergoes rapid growth and protein synthesis inpreparation for mitosis. In some embodiments, the G2/M checkpoint, alsoknown as the DNA damage checkpoint, ensures that the cell underwent allof the necessary changes during the S and G2 phases and is ready todivide.

Wee1 is a nuclear kinase belonging to the Ser/Thr family of proteinkinases. It participates in cell cycle checkpoint by inhibiting entry ofthe cell into mitosis, through inhibiting the Cdk1 protein.

As used herein, the terms “AZD1775” and “MK1775” contemplates a highlyselective, potent, ATP competitive, small molecule inhibitor of Wee1kinase. AZD1775 inhibits Wee1 activity and induces DNA damage as well asG2 checkpoint escape. In some embodiments AZD1775 or MK1775 contemplatesa molecule having the chemical structure

Embodiments of the present disclosure are further described in thefollowing examples. The examples are merely illustrative and do not inany way limit the scope of the invention as claimed.

Examples

Referring to FIGS. 1A and 1B, the inventors assessed the impact ofvarious concentrations of a G2/M checkpoint inhibitor (i.e., Wee1 kinaseinhibitor—AZD1775) on various cell lines with different geneticbackgrounds. All of the cell lines tested in FIGS. 1A and 1B where KRasmutant, some of the cell lines were p53 mutants, and some of the celllines (i.e., A427, A549, and H2030) were LKB1 deficient. All cell lineswere adenocarcinoma from patients with NSCLC. As illustrated in thesefigures, the cells responded differently to increasing concentrations ofAZD1775, with the LKB1 deficient cells having the greatest reducedviability (FIG. 1A) and lowest EC₅₀ values (FIG. 1B).

Next, referring to FIGS. 2A-2C, the LKB1 deficient lines discussed abovein reference to FIGS. 1A and 1B were assessed for percent viability inthe presence of increasing concentrations of AZD1775. Moreover, as allthree cell lines were already LKB1 deficient, the inventors wanted toelucidate the role of LKB1 in the sensitivity of these cells to AZD1775.As such, each cell line with transfected with either a rescue copy ofLKB1 (referred to as “LKB1” in these figures) or a copy of LKB1 with anon-functional kinase domain (referred to as KDLKB1 in these figures),which would produce non-functional LKB1. As illustrated in FIGS. 2A-2C,the cells transfected with the rescue copy of LKB1 were less sensitiveto AZD1775, as measured by percent viability, compared to cells thatwere transfected with the control non-functional kinase version of LKB1.

Referring now to FIG. 2D, a similar experiment was conducted on murinecells that were KRas mutant and LKB1 deficient (3381A cells). In theseexperiments, these murine cells were transfected with either a rescuecopy of LKB1 (referred to as “LKB1” in FIG. 2D) or an empty vector thatdid not include a copy of LKB1 (referred to as pBabe in FIG. 2D). Onceagain, this data shows that the cells transfected with the rescue copyof LKB1 were less sensitive to AZD1775, as measured by percentviability, compared to cells that were transfected with the controlempty vector. Taken together, the data in FIGS. 2A-2D illustrates thatcells with an LKB1 deficiency exhibit increased sensitivity to AZD1775,as shown by decreased cell viability.

Referring now to FIGS. 3A-3C, the investigators demonstrate that thecombination of AZD1775 with conventional chemotherapeutics results in asynergistic effect on the NSCLC cells. Similar to the previousexperiments, H2030 cells (KRas mutant and LKB1 deficient) weretransfected with a rescue copy of LKB1 or a version of LKB1 with anon-functional kinase domain that produces non-functional LKB1 protein.Moreover, these different cells were treated with AZD1775 (also referredto as MK1775) in combination with cisplatin (FIG. 3A), pemetrexed (FIG.3B), or gemcitabine (FIG. 3C). The data in these figures demonstratesthat AZD1775 has a negative impact on cell viability of the LKB1deficient cells and that the viability of these cells is further reducedwhen AZD1775 is combined with DNA-damaging compounds or DNArepair-inhibiting compounds. Referring to FIG. 3D, a similar phenomenonis noted in the 3381A murine cells in the case of cisplatin; however,similar data was also noted with pemetrexed and gemcitabine.

Next, referring now to FIGS. 4A and 4B, the investigators examined cellsignaling molecules in A549 and H2030 cells, including both rescue andcontrol conditions. The investigators treated with AZD1775 alone and incombination with 2Gy of ionizing radiation. As illustrated in FIGS. 4Aand 4B, the LKB1-cells exhibit increased amounts of phosphorylated AMPK,but the LKB1-deficient cells exhibit increased amount of phosphorylatedH2AX, which is a known indicator of DNA damage. As such, this datafurther illustrates that not only does the combination of AZD1775 andLKB1 deficiency lead to DNA damage and cellular injury, but the additionof a second treatment modality (radiation) can further exacerbate thisphenomenon.

Referring now to FIG. 5, the investigators looked at the timingregarding the effects of AZD1775, alone or in combination with anotherchemotherapeutic (cisplatin). A549 cells (with and without LKB1 rescuetransfections) were treated as indicated in FIG. 5 and then totalprotein was extract 4 hours after treatment and 24 hours aftertreatment. Significantly, at 24 hours post treatment, there is adiscernable increased in the levels of cleaved PARP in the A549 cellsthat remain LKB1 deficient. This cleaved PARP is an indicator of theinduction of apoptosis and efficacy of the combination treatment ofAZD1775/MK1775 and cisplatin.

This is further illustrated in FIG. 6 in which LKB1 deficient H2030cells exhibit increased Caspase 3 cleavage when treated with acombination of AZD1775 and cisplatin, with Caspase 3 cleavage beinganother indicator of the induction of apoptosis. A similar phenomenon isalso seen in FIGS. 7A and 7B in which there is increased detection ofcleaved Caspase 3 in A549 cells that are LKB1 deficient and treated witha combination of AZD175 and gemcitabine (FIG. 7A) and H2030 cells thatare LKB1 deficient and treated with the same combination (FIG. 7B).

Referring to FIG. 8, the investigators further substantiate the improvedtreatment capabilities AZD1775 in combination with otherchemotherapeutics in LKB1 deficient cells. In particular, these H2030were divided into three conditions (i) pBabe in which the cells weretransfected with an empty vector and remained LKB1 deficient; (ii) LKB1in which the cells were transfected with a functioning rescue copy ofLKB1; and (iii) p53 in which the cells were transfected with afunctioning rescue copy of p53 (i.e., the cells functioned as wild typep53 cells). The data in FIG. 8 shows that the combination of AZD1775 andcisplatin leads to significantly increased amounts of cleaved PARP andcleaved Caspase 3, which both indicate an increased amount of apoptosisoccurring in this population of H2030 cells. Moreover, cells in theother conditions (functioning LKB1 and function p53) did not exhibit theincreases in cleaved PARP and Caspase 3, although the p53 cells didexhibit increased cleavage of PARP and Caspase 3 relative to thefunctional LKB1 cells.

Referring to FIG. 9, the investigators transiently infected 6-8 week oldKRas^(G12D)/LKB1^(FL/FL)/luci with 5×10⁶P.F.U. of adenoCre viaintratracheal inhalation. Mice received scans using the Xenogen IVIS at8 weeks to identify disease. Mice were then randomized to Cisplatin(once per week, 2.5 mg/kg) or AZD1775 (30 mg/kg, three times perweek)+cisplatin and treated for four weeks. Survival was calculated vialog-rank analysis. It was observed that the median survival rate of micereceiving Cisplatin was 12 weeks, whereas the median survival rate ofmice receiving Cisplatin+AZD1775 was 14 weeks. For this set ofexperiments, the hazard ratio was 0.3972, while the confidence intervalwas 0.0605-0.6086.

It should be understood from the foregoing that, while particularembodiments have been illustrated and described, various modificationscan be made thereto without departing from the spirit and scope of theinvention as will be apparent to those skilled in the art. Such changesand modifications are within the scope and teachings of this inventionas defined in the claims appended hereto.

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein. A varietyof advantageous and disadvantageous alternatives are mentioned herein.It is to be understood that some preferred embodiments specificallyinclude one, another, or several advantageous features, while othersspecifically exclude one, another, or several disadvantageous features,while still others specifically mitigate a present disadvantageousfeature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the invention extend beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

Many variations and alternative elements have been disclosed inembodiments of the present invention. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are the selection of constituentmodules for the inventive compositions, and the diseases and otherclinical conditions that may be diagnosed, prognosed or treatedtherewith. Various embodiments of the invention can specifically includeor exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a,” “an,” and “the” and similarreferences used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans can employ such variations asappropriate, and the invention can be practiced otherwise thanspecifically described herein. Accordingly, many embodiments of thisinvention include all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that can be employed can be within thescope of the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention can be utilized inaccordance with the teachings herein. Accordingly, embodiments of thepresent invention are not limited to that precisely as shown anddescribed.

What is claimed is:
 1. A method of treating adenocarcinoma in a subject,wherein the adenocarcinoma cells are LKB1 deficient, the methodcomprising the steps of contacting the LKB1 deficient adenocarcinomacells with a therapeutically effective amount of a Wee1 kinaseinhibitor.
 2. The method of claim 1, wherein the adenocarcinoma is lungcancer.
 3. The method of claim 2, wherein the lung cancer is non-smallcell lung cancer.
 4. The method of claim 1, wherein the Wee1 kinaseinhibitor is AZD1775.
 5. The method of claim 1 and further comprisingcontacting the LKB1 deficient adenocarcinoma cells with atherapeutically effective amount of a second pharmaceutical composition.6. The method of claim 5, wherein the second pharmaceutical compositioncomprises at least one of the following: a DNA-damaging compound and aDNA repair-inhibiting compound.
 7. The method of claim 5, wherein thesecond pharmaceutical composition comprises at least one of cisplatin,carboplatin, pemetrexed, gemcitabine, and a combination thereof.
 8. Amethod of treating adenocarcinoma cells in a subject, the methodcomprising the steps of: receiving a sample of the adenocarcinoma cells;determining if at least a portion of the sample of the adenocarcinomacells is LKB1 deficient; and contacting the adenocarcinoma cells with atherapeutically effective amount of a Wee1 kinase inhibitor.
 9. Themethod of claim 8, wherein the adenocarcinoma is lung cancer.
 10. Themethod of claim 9, wherein the lung cancer is non-small cell lungcancer.
 11. The method of claim 8, wherein the Wee1 kinase inhibitor isAZD1775.
 12. The method of claim 8 and further comprising contacting theadenocarcinoma cells that are LKB1 deficient with a therapeuticallyeffective amount of a second pharmaceutical composition, wherein thetherapeutically effective amount of the second pharmaceuticalcomposition is less than an amount that would be used without the Wee1kinase inhibitor.
 13. The method of claim 12, wherein the secondpharmaceutical composition comprises at least one of the following: aDNA-damaging compound and a DNA repair-inhibiting compound.
 14. Themethod of claim 8 and further comprising determining if at least aportion of the sample of the adenocarcinoma cells is KRAS mutant.
 15. Amethod of treating adenocarcinoma cells in a subject, wherein theadenocarcinoma cells are LKB1 and KRAS mutant, the method comprising thesteps of contacting the LKB1 deficient and KRAS mutant adenocarcinomacells with a therapeutically effective amount of a Wee1 inhibitor; andcontacting the LKB1 deficient and KRAS mutant adenocarcinoma cancercells with a therapeutically effective amount of a second pharmaceuticalcomposition.
 16. The method of claim 15, wherein the adenocarcinoma islung cancer.
 17. The method of claim 16, wherein the lung cancer isnon-small cell lung cancer.
 18. The method of claim 15, wherein the Wee1kinase inhibitor is AZD1775.
 19. The method of claim 15, wherein thesecond pharmaceutical composition comprises at least one of cisplatin,carboplatin, pemetrexed, gemcitabine, and a combination thereof.
 20. Themethod of claim 15, wherein the LKB1 deficient and KRAS mutantadenocarcinoma cells are sensitive to treatment with the secondpharmaceutical composition.